Histone deacetylase as target for antiprotozoal agents

ABSTRACT

Histone deacetylase inhibition provides a target for identifying potential antiprotozoal compounds. Histone deacetylase inhibitors are useful as therapeutic agents against protozoal infections.

This is a division of application Ser. No. 08/716,978 filed Sep. 20,1996.

BACKGROUND OF THE INVENTION

Parasitic protozoa are responsible for a wide variety of infections inman and animals. Many of the diseases are life threatening to the hostand cause considerable economic loss in animal husbandry. For example,malaria remains a significant health threat to humans despite massiveinternational attempts to eradicate the disease; trypanosomiasis such asChagas disease caused by Trypanosoma cruzi and African sleeping sicknesscaused by T. brucei are not uncommon in Africa and South America; andopportunistic infections in immunocompromised hosts caused byPneumocystis carinii, Toxoplasma gondii, Cryptosporidium sp. arebecoming increasingly significant in the developed countries.

A protozoal infection of great economic importance is coccidiosis, awidespread disease of domesticated animals produced by infections byprotozoa of the genus Eimeria. Some of the most significant of Eimeriaspecies are those in poultry namely E. tenella, E. acervulina, E.necatrix, E. brunetti and E. maxima. The disease is responsible for highlevels of morbidity and mortality in poultry and can result in extremeeconomic losses.

In some protozoal diseases, such as Chagas disease, there is nosatisfactory treatment; in others, drug-resistant strains of theprotozoa may develop. Accordingly, there exists a continued need toidentify new and effective anti-protozoal drugs. However, antiparasiticdrug discovery has been, for the most part, a random and laboriousprocess through biological screening of natural products and syntheticcompounds against a panel of parasites. This process can be greatlyfacilitated and made more specific if a target of antiprotozoal drugscan be identified, and incorporated into the screening process.

Histone deacetylase and histone acetyltransferase together control thenet level of acetylation of histones. Inhibition of the action ofhistone deacetylase results in the accumulation of hyperacetylatedhistones, which in turn is implicated in a variety of cellularresponses, including altered gene expression, cell differentiation andcell-cycle arrest. Recently, trichostatin A and trapoxin A have beenreported as reversible and irreversible inhibitors, respectively, ofmammalian histone deacetylase (see e.g., Yoshida et al, Bioassays, 1995,17(5):423-430). Trichostatin A has also been reported to inhibitpartially purified yeast histone deacetylase (Sanchez del Pino et al,Biochem. J., 1994, 303:723-729). Trichostatin A is an antifungalantibiotic and has been shown to have anti-trichomonal activity as wellas cell differentiating activity in murine erythroleukemia cells, andthe ability to induce phenotypic reversion in sis-transformed fibroblastcells (see e.g. U.S. Pat. No. 4,218,478; Yoshida et al, Bioassays, 1995,17(5):423-430 and references cited therein). Trapoxin A, a cyclictetrapeptide, induces morphological reversion of v-sis-transformedNIH3T3 cells (Yoshida and Sugita, Jap. J. Cancer Res., 1992,83(4):324-328). The present inventors have found that a number of cyclictetrapeptides structurally related to trapoxin A are inhibitors ofhistone deacetylase, and also possess antiprotozoal activity.

SUMMARY OF THE INVENTION

The present invention relates to histone deacetylase as a target forantiprotozoal agents. More particularly, the invention concerns a methodfor identifying potential antiprotozoal agents by determining whether atest compound is capable of inhibiting the action of histonedeacetylase. The invention also concerns a method for treating protozoalinfections by administering to a host suffering from protozoal infectiona therapeutically effective amount of a compound that inhibits histonedeacetylase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows histone deacetylase inhibitors cause hyperacetylation ofHeLa cell histones.

FIG. 2 shows histone deacetylase inhibitors cause hyperacetylation ofhistones from P. falciparum.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the present invention provides a method for identifyingcompounds having antiprotozoal activity comprising:

(a) contacting a histone deacetylase, or an extract containing histonedeacetylase with (i) a known amount of a labeled compound that interactswith a histone deacetylase; and (ii) a known dilution of a test compoundor a natural product extract; and

(b) quantitating the percent inhibition of interaction of said labeledcompound induced by said test compound.

In another aspect the present invention provides a method foridentifying compounds having antiprotozoal activity comprising:

(a) contacting an intact host or protozoal cell with a test compound ora natural product extract;

(b) disrupting said cell to obtain histones; and

(c) determining the level of histone acetylation.

The methods of the invention provides a facile and specific assay toscreen compounds as potential antiprotozoal drugs.

In the present invention the histone deacetylase (also referred toherein as HDA or HDAase) may be a purified or partially purified nativeenzyme, a cloned histone deacetylase or an engineered variant thereof, acrude preparation of the enzyme, or an extract containing histonedeacetylase activity. The enzyme may be from a mammalian (e.g. humancervical carcinoma, HeLa cell), avian (e.g. chicken liver or erythrocytenuclei) or protozoal (e.g. Eimeria tenella or P. berghei) source;preferably a protozoal histone deacetylase is used. Fragments of histonedeacetylase that retain the desired enzyme activity is also within thescope of the invention.

A compound that interacts with histone deacetylase may be one that is asubstrate for the enzyme, one that binds the enzyme at its active site,or one that otherwise acts to alter enzyme activity by binding to analternate site. A substrate may be acetylated histones, or a labeledacetylated peptide fragment derived therefrom such asAcGly-Ala-Lys(ε-Ac)-Arg-His-Arg-Lys(ε-Ac)-ValNH₂, or other synthetic ornaturally occuring substrates. Examples of compounds that bind tohistone deacetylase are known inhibitors such as n-butyrate,trichostatin, trapoxin A, as well as other inhibitors described herein.The compound that interacts with histone deacetylase is preferablylabeled to allow easy quantitation of the level of interaction betweenthe compound and the enzyme. A prefered radiolabel is tritium.

The test compound may be a synthetic compound, a purified preparation,crude preparation, or an initial extract of a natural product obtainedfrom plant, microorganism or animal sources.

One embodiment of the present method is based on test compound inducedinhibition of histone deacetylase activity. The enzyme inhibition assayinvolves adding histone deacetylase or an extract containing histonedeacetylase to mixtures of an enzyme substrate and the test compound,both of which are present in known concentrations. The amount of theenzyme is chosen such that ≦20% of the substrate is consumed during theassay. The assay is carried out with the test compound at a series ofdifferent dilution levels. After a period of incubation, the labeledportion of the substrate released by enzymatic action is separated andcounted. The assay is generally carried out in parallel with a control(no test compound) and a positive control (containing a known enzymeinhibitor instead of a test compound). The concentration of the testcompound at which 50% of the enzyme activity is inhibited (IC₅₀) isdetermined using art recognized method.

Although enzyme inhibition is the most direct measure of the inhibitoryactivity of the test compound, the present inventors have found thatresults obtained from competitive binding assay in which the testcompound competes with a known inhibitor for binding to the enzymeactive site correlate well with the results obtained from enzymeinhibition assay described above. The binding assay represents a moreconvenient way to assess enzyme inhibition since it allows the use of acrude extract containing histone deacetylase rather than partiallypurified enzyme. The use of a crude extract may not always be suitablein the enzyme inhibition assay because other enzymes present in theextract may act on the histone deacetylase substrate. The competitionbinding assay is carried out by adding the histone deacetylase or anextract containing histone deacetylase activity to a mixture of the testcompound and a labeled inhibitor, both of which are present in themixture in known concentrations. After incubation, the enzyme-inhibitorcomplex is separated from the unbound labeled inhibitors and unlabeledtest compound, and counted. The concentration of the test compoundrequired to inhibit 50% of the binding of the labeled inhibitor to thehistone deacetylase (IC₅₀) is calculated.

In a preferred embodiment, the method of the present invention utilizesa histone deacetylase or an extract containing histone deacetylaseobtained from a protozoal source, such as Eimeria or Plasmodium sp.

In a more preferred embodiment, the method of the present inventionfurther comprises determining the IC₅₀ of test compounds against hosthistone deacetylase in either the enzyme inhibition assay or the bindingassay as described above, to identify those compounds that haveselectivity for parasitic histone deacetylase over that of a host. Theassays are the same as previously described, with the histonedeacetylase activity obtained from a host of protozoa; for example thehost histone deacetylase may be obtained from a mammalian source, e.g.human, or an avian source, e.g. chicken.

Another method useful to identify inhibitors that are selective forparasitic histone deacetylase is the use of acid urea trion (AUT) gelelectrophoresis to determine the level of acetylation of histones. Thuscompounds that cause hyperacetylation of parasitic histone with no orlittle hyperacetylation of host histone would be considered selectiveparasitic histone deacetylase inhibitors.

Where the enzyme inhibition or binding assay utilizes a crudepreparation or an extract containing histone deacetylase, the target ofthe test compound may be verified by examining the level of histoneacetylation. Thus, the intact host or parasitic cell containing theenzyme is treated with the test compound. Alternatively, intact host orparasite cells containing the enzyme is treated with test compound inthe presence of labeled sodium acetate (¹⁴C is the preferred label). Inboth cases the cells are lysed, histones are partially purified, andanalyzed by acid urea triton (AUT) gel electrophoresis. Proteins aredetected by staining or by detection of radiolabel by fluorography.Differentially acetylated species can readily be distinguished on suchAUT gels due to the slower migration of acetylated species. A histonedeacetylase inhibitor will cause hyperacetylation of histones. Since theAUT gel electrophoresis uses intact cells treated with the testcompound, this techniques may also be used to identify prodrugs that maybe converted to histone deacetylase inhibitor within the cellularenvironment, but may not be so identified by assay based on the enzymeitself.

In another aspect the present invention provides a method for thetreatment of protozoal infections comprising administering to a hostsuffering from a protozoal infection a therapeutically effective amountof a compound which inhibits histone deacetylase. A therapeuticallyeffective amount may be one that is sufficient to inhibit histonedeacetylase of the causative protozoa.

Examples of known compounds which may be histone deacetylase inhibitorsand therefore useful in the treatment of protozoal diseases include, butare not limited to, trichostatin A, trapoxin A, HC-toxin, chlamydocin,Cly-2, WF-3161, Tan-1746, apicidin and analogs thereof. Trichostatin A,trapoxin A, HC-toxin, chlamydocin, Cy-2, and WF-3161, as well asderivatives thereof are well known in the art. HC-Toxin is described inLiesch et al. (1982) Tetrahedron 38 45-48; Trapoxin A is described inItazaki et al. (1990) J. Antibiot. 43, 1524-1532; WF-3161 is describedin Umehana et al. (1983) J. Antibiot. 36, 478-483; Cly-2 is described inHirota et al (1973) Agri. Biol. Chem 37, 955-56; Chlamydocin isdescribed in Closse et al. (1974) Helv. Chim. Acta 57, 533-545 and Tan1746 is described in Japanese Patent No. 7196686 to Takeda Yakuhin KogyoKK.

Apicidin and analogs thereof referred to herein have the followingstructural formulae:

Apicidin Ia, Ib, Ic are described in pending applications U.S. Ser. No.08/281,325 filed Jul. 27, 1994 and 08/447,664 filed May 23, 1995. Theyare produced from a strain of Fusarium as disclosed in theabovementioned applications.

Histone deacetylase inhibitors are useful as antiprotozoal agents. Assuch, they may be used in the treatment and prevention of protozoaldiseases in human and animals, including poultry. Examples of protozoaldiseases against which histone deacetylase inhibitors may be used, andtheir respective causative pathogens, include: 1) amoebiasis(Dientamoeba sp., Entamoeba histolytica); 2) giardiasis (Giardialamblia); 3) malaria (Plasmodium species including P. vivax, P.falciparum, P. malariae and P. ovale); 4) leishmaniasis (Leishmaniaspecies including L. donovani, L. tropica, L. mexicana, and L.braziliensis); 5) trypanosomiasis and Chagas disease (Trypanosomaspecies including T. brucei, T. theileri, T. rhodesiense, T. gambiense,T. evansi, T. equiperdum, T. equinum, T. congolense, T. vivax and T.cruzi); 6) toxoplasmosis (Toxoplasma gondii); 7) neosporosis (Neosporacaninum); 8) babesiosis (Babesia sp.); 9) cryptosporidiosis(Cryptosporidium sp.); 10) dysentary (Balantidium coli); 11) vaginitis(Trichomonas species including T. vaginitis, and T. foetus); 12)coccidiosis (Eimeria species including E. tenella, E. necatrix, E.acervulina, E. maxima and E. brunetti, E. mitis, E. bovis, E.melagramatis, and Isospora sp.); 13) enterohepatitis (Histomonasgallinarum), and 14) infections caused by Anaplasma sp., Besnoitia sp.,Leucocytozoan sp., Microsporidia sp., Sarcocystis sp., Theileria sp.,and Pneumocystis carinii.

Histone deacetylase inhibitors are preferably used in the treatment orprevention of protozoal infections caused by a member of the sub-phyllumApicomplexans. More preferably histone deacetylase inhibitors arepreferably used in the treatment or prevention of malaria,toxoplasmosis, cryptosporidiosis and trypanosomiasis in humans andanimals; and in the management of coccidiosis, particularly in poultry,either to treat coccidial infection or to prevent the occurrence of suchinfection.

In the case that a histone deacetylase inhibitor is expected to beadministered on a chronic basis, such as in the prevention ofcoccidiosis in poultry, the histone deacetylase inhibitor preferably isselective for protozoal over the host histone deacetylase. Long termadministration of such a selective inhibitor would minimize adverseeffects to the host due to histone deacetylase inhibition.

Two specific examples of using histone deacetylase inhibitors to preventthe establishment of parasitic infections in humans and animals are 1)the prevention of Plasmodium (malaria) infection in humans in endemicareas and 2) the prevention of coccidiosis in poultry by administeringthe compound continuously in the feed or drinking water. Malaria is thenumber one cause of death in the world. The disease is transmitted bymosquitoes in endemic areas and can very rapidly progress to a lifethreatening infection. Therefore, individuals living in or visitingareas where malaria carrying mosquitoes are present routinely takeprophylactic drugs to prevent infection. The histone deacetylaseinhibitor would be administered orally or parenterally one or moretime(s) a day. The dose would range from 0.01 mg/kg to 100 mg/kg. Thecompound could be administered for the entire period during which thepatient or animal is at risk of acquiring a parasitic infection.

Coccidiosis is a disease which can occur in humans and animals and iscaused by several genera of coccidia. The most economically importantoccurrence of coccidiosis is the disease in poultry. Coccidiosis inpoultry is caused by protozoan parasites of the genus Eimeria. Thedisease can spread quite rapidly throughout flocks of birds viacontaminated feces. The parasites destroy gut tissue and thereforedamage the gut lining impairing nutrient absorption. An outbreak ofcoccidiosis in a poultry house can cause such dramatic economic lossesfor poultry producers that it has become standard practice to useanticoccidial agents prophylactically in the feed. A histone deacetylaseinhibitor would be administered in the feed or drinking water for, aportion of, or the entire life of the birds. The dose would rangebetween 0.1 ppm to 500 ppm in the feed or water.

For treatment of established parasitic infections in humans or animals,the histone deacetylase inhibitor could be administered orally orparenterally once the infection is suspected or diagnosed. The treatmentperiod would vary according to the specific parasitic disease and theseverity of the infection. In general the treatment would be continueduntil the parasites were eradicated and/or the symptoms of the diseasewere resolved. Two specific examples are the treatment of a 1)Cryptosporidium parvum infection in an animal or human and treatment ofacute Plasmodium falciparum malaria in humans. Cryptosporidium parvum isa protozoan parasite that infects and destroys cells lining theintestinal tract of humans and animals. The infection establishes quiterapidly and has acute effects on the patient. In the case of humans,patients get severe dysentery for a period of 5-7 days. In immunecompromised patients C. parvum infections can persist and can be lifethreatening. In animals C. parvum infection is the number one cause ofdeath in young dairy calves. A C. parvum infection can be easilydiagnosed by symptoms and examination of a stool sample. Once thedisease is suspected and/or diagnosed treatment with a histonedeacetylase inhibitor can be initiated. The dose would vary between 0.01mg/kg to 500 mg/kg. Treatments would be one or more time(s) a day,orally or parenterally until the infection is eliminated. Routinely thisdosing period would be 1-3 weeks.

P. falciparum causes acute life threatening malarial infections inhumans. The infection if left untreated can quite often result in deathof the patient. A malaria infection can be easily diagnosed by symptomsand examination of a blood sample from the patient. Treatment would beinitiated following diagnosis. A histone deacetylase inhibitor would beadministered one or more time(s) a day, orally or parenterally, untilthe infection was eliminated. The dose would range between 0.01 mg/kg to200 mg/kg.

Histone deacetylase inhibitors may be administered to a host in need oftreatment in a manner similar to that used for other antiprotozoalagents; for example, they may be administered parenterally, orally,topically, or rectally. The dosage to be administered will varyaccording to the particular compound used, the infectious organisminvolved, the particular host, the severity of the disease, physicalcondition of the host, and the selected route of administration; theappropriate dosage can be readily determined by a person skilled in theart. For the treatment of protozoal diseases in human and animals, thedosage may range from 0.01 mg/kg to 500 mg/kg. For prophylactic use inhuman and animals, the dosage may range from 0.011 mg/kg to 100 mg/kg.For use as an anticoccidial agent, particularly in poultry, the compoundis preferably administered in the animals' feed or drinking water. Thedosage ranges from 0.1 ppm to 500 ppm.

The compositions of the present invention comprises a histonedeacetylase inhibitor and an inert carrier. The compositions may be inthe form of pharmaceutical compositions for human and veterinary usage,or in the form of feed composition for the control of coccidiosis inpoultry. The term “composition” is intended to encompass a productcomprising the active ingredient(s), and the inert ingredient(s) thatmake up the carrier, as well as any product which results, directly orindirectly, from combination, complexation or aggregation of any two ormore of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions of one or more of theingredients. The composition of the present invention thus includes acomposition when made by admixing a histone deacetylase inhibitor andinert carrier.

The pharmaceutical compositions of the present invention comprise ahistone deacetylase inhibitor as an active ingredient, and may alsocontain a pharmaceutically acceptable carrier and optionally othertherapeutic ingredients. The compositions include compositions suitablefor oral, rectal, topical, and parenteral (including subcutaneous,intramuscular, and intravenous) administrations, although the mostsuitable route in any given case will depend on the particular host, andnature and severity of the conditions for which the active ingredient isbeing administered. The pharmaceutical compositions may be convenientlypresented in unit dosage form and prepared by any of the methodswell-known in the art of pharmacy.

In practical use, a histone deacetylase inhibitor can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous).

In preparing the compositions for oral dosage form, any of the usualpharmaceutical media may be employed. For example, in the case of oralliquid preparations such as suspensions, elixirs and solutions, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like may be used; or in the case of oral solidpreparations such as powders, capsules and tablets, carriers such asstarches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, disintegrating agents, and the like may beincluded. Because of their ease of administration, tablets and capsulesrepresent the most advantageous oral dosage unit form in which casesolid pharmaceutical carriers are obviously employed. If desired,tablets may be coated by standard aqueous or nonaqueous techniques. Inaddition to the common dosage forms set out above, histone deacetylaseinhibitors may also be administered by controlled release means and/ordelivery devices.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient, as a powder or granules or as a solution or a suspension inan aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or awater-in-oil liquid emulsion. Such compositions may be prepared by anyof the methods of pharmacy but all methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. For example, a tablet may be prepared by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, theactive ingredient in a free-flowing form such as powder or granules,optionally mixed with a binder, lubricant, inert diluent, surface activeor dispersing agent. Molded tablets may be made by molding in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. Desirably, each tablet contains from about 1 mg to about500 mg of the active ingredient and each cachet or capsule contains fromabout 1 to about 500 mg of the active ingredient.

Pharmaceutical compositions of the present invention suitable forparenteral administration may be prepared as solutions or suspensions ofthese active compounds in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g. glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Suitable topical formulations include transdermal devices, aerosols,creams, ointments, lotions, dusting powders, and the like. Theseformulations may be prepared via conventional methods containing theactive ingredient. To illustrate, a cream or ointment is prepared bymixing sufficient quantities of hydrophilic material and water,containing from about 5-10% by weight of the compound, in sufficientquantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the combination with the softened ormelted carrier(s) followed by chilling and shaping moulds.

It should be understood that in addition to the aforementioned carrieringredients the pharmaceutical formulations described above may include,as appropriate, one or more additional carrier ingredients such asdiluents, buffers, flavoring agents, binders, surface-active agents,thickeners, lubricants, preservatives (including anti-oxidants) and thelike, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

For use in the management of coccidiosis in poultry, a histonedeacetylase inhibitor may be conveniently administered as a component ofa feed composition. Suitable poultry feed composition will typicallycontain from about 1 ppm to about 1000 ppm, preferably from about 0.01%to about 0.1% percent, by weight of a histone deacetylase inhibitor. Theoptimum levels will naturally vary with the species of Eimeria involved,and can be readily determined by one skilled in the art. Levels of inpoultry feed of from about 0.01% to about 0.1% percent by weight of thediet are especially useful in controlling the pathology associated withE. tenella, while the preferred concentration for similar control ofintestinal-dwelling species is from about 0.01% to about 0.1% percent byweight of the diet. Amounts of about 0.01% to about 0.1% percent byweight are advantageous in reducing the pathogenic effects of both cecaland intestinal coccidiosis.

In the preparation of poultry feed, a histone deacetylase inhibitor maybe readily dispersed by mechanically mixing the same in finely groundform with the poultry feedstuff, or with an intermediate formulation(premix) that is subsequently blended with other components to preparethe final poultry feedstuff that is fed to the poultry. Typicalcomponents of poultry feedstuffs include molasses, fermentationresidues, corn meal, ground and rolled oats, wheat shorts and middlings,alfalfa, clover and meat scraps, together with mineral supplements suchas bone meal, calcium carbonate and vitamins.

Compositions containing a compound of formula I may also be prepared inpowder or liquid concentrate form. In accordance with standardveterinary formulation practice, conventional water soluble excipients,such as lactose or sucrose, may be incorporated in the powders toimprove their physical properties. Thus particularly suitable powders ofthis invention comprise 50 to 100% w/w, and preferably 60 to 80% w/w ofthe combination and 0 to 50% w/w and preferably 20 to 40% w/w ofconventional veterinary excipients. These powders may either be added toanimal feedstuffs, for example by way of an intermediate premix, ordiluted in animal drinking water.

Liquid concentrates of this invention suitably contain a water-solublecompound combination and may optionally include a veterinarilyacceptable water miscible solvent, for example polyethylene glycol,propylene glycol, glycerol, glycerol formal or such a solvent mixed withup to 30% v/v of ethanol. The liquid concentrates may be administered tothe drinking water of animals, particularly poultry.

Preparation of Materials Used in the Examples

1. Apicidin (Ia)

(a) Seed Culture of Fusarium sp. ATCC 74322

A portion of agar slant containing Fusarium sp. ATCC 74322 wasaseptically transferred to a 250·mL unbaffled flask containing 50 mL ofthe seed medium having the following composition:

Yeast extract (Difco) 4.0 g/L Malt extract (Difco) 8.0 g/L Glucose 4.0g/L Junlon (polyacrylic acid, 1.5 g/L Kouyok Trading Co., Tokyo)distilled water q.v. 1 L pH adjusted to 7.0 prior to sterilization.

The culture was incubated on a 2-inch throw gyratory shaker, (220 rpm)for 3 days at 25° C., 85% relative humidity (rh), to obtain biomass.Portions of the biomass were transferred into sterile vials containingglycerol and frozen (as frozen vegetative mycelia). These weremaintained in a final concentration of 10-15% glycerol at −75° C.

A vial of the above frozen vegetative mycelia was thawed to roomtemperature and used to inoculate the seed medium (1 mL per 50 mL seedmedium), and the culture was incubated for 2-4 days under the conditionsdescribed above. Seed medium of the following composition may also beused to prepare the seed culture:

Corn steep liquor 5.0 g/L Tomato paste 40.0 g/L Oat flour 10.0 g/LGlucose 10.0 g/L Trace element mix* 10 mL/L distilled water q.v. 1 L pHadjusted to 6.8 prior to sterilization. *composition (g/L) = FeSO₄ ·7H₂O (1.0); MnSO₄ · 4H₂O (1.0); CuCl₂ · H₂O (0.025); CaCl₂ (0.1); H₃BO₃(0.056); (NH₄)₆MoO₂ · 4H₂O (0.019); ZnSO₄ · 7H₂O (0.2).

(b) Production Culture of Fusarium sp.

An aliquot of the seed culture (12 mL) was placed into 220 mL of theliquid portion of the production medium having the followingcomposition:

Glucose 150.0 g/L NZ amine Type A 4.0 g/L (Sheffield Products) Urea 4.0g/L K₂HPO₄ 0.5 g/L KCl 0.25 g/L MgSO₄ · 7H₂O 0.25 g/L ZnSO₄ · 7H₂O 0.9g/L CaCO₃ 16.5 g/L distilled water q.v. 1 L (pH 7.0)

This mixture was swirled vigorously to disperse the biomass and thenpoured into a 2-liter roller culture vessel which contained 675 cubiccentimeters of steam-sterilized large-particle vermiculite (the solidportion). The contents of the roller bottle were shaken/mixed to insurehomogeneous inoculation and coverage. The roller bottles were incubatedhorizontally, revolving at approximately 4 rpm on a Wheaton rollerapparatus, at 22° C., 75% rh for 24 days, to obtain secondary metaboliteproduction in the fermentation medium.

The culture also produced the desired metabolites in liquid medium (50mL in 250 mL unbaffled flask) containing the liquid portion of the aboveproduction medium. The flasks were incubated at 22° C., 75% rh for 7-14days.

Fermentation in 23 L tank fermentor was also carried out using the aboveliquid production medium. Thus, 10 L of the production medium plus 10 mLof Polyglycol 2000 (Dow) was inoculated with 500 mL of the seed culture,and the fermentation carried out at 21° C., with agitation set at 400rpm, air flow at 5 L/min and pressure at 0.3 bar. The agitation wasincreased to 500 and then 600 rpm, and air flow to 10 L/min on day 6 offermentation (day 0 being the day fermentation started). Thefermentation broth was harvested after 14 days for product isolation.The same conditions were also used in 23 L tank fermentation using theproduction medium whose composition is as follows:

Glucose 150.0 g/L Fructose 15.0 g/L Sucrose 40.0 g/L NZ amine Type E 4.0g/L (Sheffield Products) Urea 4.0 g/L K₂HPO₄ 0.5 g/L KCl 0.25 g/L MgSO₄· 7H₂O 0.25 g/L ZnSO₄ · 7H₂O 0.9 g/L CaCO₃ 8.0 g/L distilled water q.v.1 L (pH 7.0)

(c) Isolation and Purification of Apicidin

The fermentation broth (1.6 L) prepared above was extracted with methylethyl ketone (MEK). After evaporating the solvent, the residual aqueoussuspension was freeze-dried. The resulting solid mass was extensivelytriturated with methylene chloride and filtered. The solution was firstfractionated at high speed on a 100 ml bed of dry E. Merck silicagel 60using methylene chloride-methanol 97:3 (v/v). This afforded an enrichedpreparation of Compound Ia after 0.6 column volumes of washing. Finalpurification could be achieved by column chromatography on EM silica gelusing ethyl acetate in methylene chloride as eluent (1:2 v/v). Yield:approx. 5 mg of homogeneous material.

2. Apicidin analogs Ib and Ic

(a) Seed Culture of Fusarium pallidoroseum

A portion of agar slant containing Fusarium pallidoroseum ATCC 74289 wasaseptically transferred to a 250·mL unbaffled flask containing 50 mL ofseed medium having the following composition:

Yeast extract (Difco) 4.0 g/L Malt extract (Difco) 8.0 g/L Glucose 4.0g/L Junlon (polyacrylic acid, 1.5 g/L Kouyok Trading Co., Tokyo)distilled water q.v. 1 L pH adjusted to 7.0 prior to sterilization.

The culture was incubated on a 2-inch throw gyratory shaker, (220 rpm)for 2 days at 25° C., 85% relative humidity (rh), to obtain biomass.Portions of the biomass were transferred into sterile vials containingglycerol and frozen (as frozen vegetative mycelia). These weremaintained in a final concentration of 10-15% glycerol at −75° C.

A vial of the above frozen vegetative mycelia was thawed to roomtemperature and used to inoculate the seed medium (1 mL per 50 mL seedmedium), and the culture was incubated under the conditions describedabove.

(b) Production Culture of Fusarium pallidoroseum

An aliquot of the seed culture (12 mL, in the above seed medium) wasplaced into 220 mL of the liquid portion of the production medium havingthe following composition:

Glucose 150.0 g/L Fructose 15.0 g/L Sucrose 40.0 g/L NZ amine Type E 4.0g/L (Sheffield Products) Urea 4.0 g/L K₂HPO₄ 0.5 g/L KCl 0.25 g/L MgSO₄· 7H₂O 0.25 g/L ZnSO₄ · 7H₂O 0.9 g/L CaCO₃ 8.0 g/L distilled water q.v.1 L (pH 7.0)

This mixture was swirled vigorously to disperse the biomass and thenpoured into a 2-liter roller culture vessel which contained 675 cubiccentimeters of steam-sterilized large-particle vermiculite (the solidportion). The contents of the roller bottle were shaken/mixed to insurehomogeneous inoculation and coverage. The roller bottles were incubatedhorizontally, revolving at approximately 4 rpm on a Wheaton rollerapparatus, at 22° C., 75% rh for 19 days, to obtain secondary metaboliteproduction in the fermentation medium.

(c) Isolation and Purification

Fermentation broth (5.6 liters), as prepared above, containing 225 mg ofcompound Ia, was extracted with methylethyl ketone by stirring for 2hours and filtering off the spent biomass. After evaporating the solventunder reduced pressure and freeze-drying the resulting aqueoussuspension, the semi-solid residue obtained was taken up in 200 ml ofmethylene chloride and filtered to remove large amounts of insoluble,unrelated materials.

The solution was again evaporated down and the new residue dissolved inmethanol. Fractionation proceeded by gel filtration on a 950 cc columnof Sephadex® LH-20 in methanol, whereupon compound Ia and severalrelated analogs eluted after 0.55-0.65 column volumes. There followed aflash chromatography step on a 120 cc E. Merck silica gel 60 columneluted with a step gradient of methanol in methylene chloride. Thetarget compounds again eluted together, in fractions corresponding tothe 3% methanol washings.

Material from the first silica gel column was put through a new 120 ccE. Merck silica gel 60 column, this time eluting with a methylenechloride-ethyl acetate 3:1 v/v mixture, and gradually increasing theethyl acetate content to 100%. This process afforded four fractions, inorder of elution,

Fraction III contained a mixture of compounds including Ib, and wasdirectly processed by HPLC (same conditions as above). Homogenouscompound Ib was found after 15.5 minutes into the gradient.

Fraction IV, on the other hand, was judged to be still too complex forsuccessful HPLC. Accordingly, it was first further enriched by gelfiltration on Sephadex® LH-20 before final purification of compound Icby HPLC (9×250 mm Zorbax® R×C₈ column, maintained at 50° C., elutedisocratically for 1 hour with 2 ml/minute 30% aqueous acetonitrile,followed by a 2-hour gradient to 60% acetonitrile). Ve=6.9-7.2 columnvolumes.

3. Apicidin Analogs IIA and IIB

A solution containing 7.2 mg Cu^((II))(OAc)₂ in 3 mL methylene chlorideand 0.15 mL pyridine under an oxygen atmosphere was prepared. To thiswas added a solution of 50 mg apicidin in 3 mL methylene chloride in oneportion. The solution was stirred at RT for 12 hours and then heated for4 hours to 45° C. The solvents were removed under reduced pressure andthe residue dissolved in 6 mL 1:1 methanol/water to which was added 150mg NaIO₄. the solution was stirred at 40° C. for 48 hours. The solutionwas diluted with brine, extracted with methylene chloride, the organiclayer dried (Na₂SO₄), filtered and concentrated under reduced pressure.The crude was partially purified by preparative thin layerchromatography (3×1000 μm silica gel plates) using 1:1 acetone/hexanesas eluant. The lower band was re-purified by preparative TLC (1×1000 μmsilica gel plate) using 1:1 acetone/hexanes as eluant yielding CompoundIIA (3 mg) and Compound IIB (2 mg). Both compounds gave satisfactoryproton NMR spectra.

Physical Properties:

Compound IIA:

R_(f)=0.33 by TLC in 1:1 acetone/hexanes

T_(r)=6.1 min in 50:50 acetonitrile/water, 2 mL/min, 4.6×30 cm Zorbax®

RX-8 column

MS: 638 (M+1)

Compound IIB:

R_(f)=0.17 by TLC in 1:1 acetone/hexanes

T_(r)=5.7 min in 50:50 acetonitrile/water, 2 mL/min, 4.6×30 cm Zorbax®

RX-8 column

MS: 608 (M+1)

3.2-³H-N-desmethoxyapicidin

Apicidin (25 mg, 0.0401 mmol) was dissolved in sieved (3:2)CH₂Cl₂/1-butanol (4 mL) and 20% palladium hydroxide on carbon (ca. 2.5mg) was added. The system was vacuum-purged thrice with hydrogen gas andthe reaction mixture stirred vigorously under 1 atm H₂ for 2 h at 23° C.(TLC control; SiO₂, 1:1 acetone-hexane; apicidin R_(f) 0.50,N-desmethoxyapicidin R_(f) 0.38). The reaction mixture was then filteredthrough a celite plug, washed thoroughly with methylene chloride and thefiltrate concentrated in vacuo. The residue was purified by flash columnchromatography (SiO₂, 1:3:96 NH₄OH—MeOH—CHCl₃, 3×9 cm) and concentratedin vacuo to provide N-desmethoxyapicidin (22.2 mg, 23.8 mg theoretical,93%) as a white powder: Analytical RP-HPLC (gradient elution of 40%acetonitrile-water to acetonitrile at 2 mL/min, peak elution at 8.5 min;Zorbax® Rx-C8, 4.6 mm×25 cm); ¹H NMR (CDCl₃, 500 MHz) satisfactory;ESI-MS (MeCN—H₂O-TFA) m/e 594 (M⁺, C₃₃H₄₇N₅O₅ requires 593.77).

N-Desmethoxyapicidin (10 mg, 0.0168 mmol) was dissolved in sieved CHCl₃(170 μL) and cooled to 0° C. Pyridinium bromide perbromide (8.1 mg,0.0253 mmol, 1.5 equiv) was added neat at 0° C. and the reaction mixturewas stirred for 45 minutes at 0° C. under an atmosphere of nitrogen (TLCcontrol; SiO₂, 1:3:96 NH₄OH—MeOH—CHCl₃; N-desmethoxyapicidin R_(f) 0.40,2-bromo-N-desmethoxyapicidin R_(f) 0.44). At 0° C. the reaction mixturewas quenched with saturated aqueous NaHCO₃ and extracted with CH₂Cl₂.The organic partition was washed with water, dried over Na₂SO₄ andconcentrated in vacuo. The crude residue was purified by preparativereverse phase HPLC (gradient elution of 40% acetonitrile-water toacetonitrile at 20 mL/min, peak elution at 22 min) and lyophilized toprovide 2-bromo-N-desmethoxyapicidin (4.3 mg, 11 mg theoretical, 40%) asa white powder: Analytical RP-HPLC (gradient elution of 40%acetonitrile-water to acetonitrile at 2 mL/min, peak elution at 9 min;Zorbax® Rx-C8, 4.6 mm×25 cm); ¹H NMR (CDCl₃, 500 MHz) satisfactory;ESI-MS (MeCN—H₂O—TFA) m/e 674 (M⁺, C₃₃H₄₆N₅O₅Br requires 672.67).

2-Bromo-N-desmethoxyapicidin (2 mg, 0.0030 mmol) was dissolved in sievedCH₂Cl₂ (1 mL) and 20% palladium hydroxide on carbon (ca. 0.5 mg) wasadded. The system was vacuum-purged twice with deuterium gas and thereaction mixture stirred vigorously under 1 atm D₂ for 2 h at 23° C.(TLC control; SiO₂, 1:3:96 NH₄OH—MeOH—CHCl₃;2-bromo-N-desmethoxyapicidin R_(f) 0.40, 2-D-N-desmethoxyapicidin R_(f)0.34). The reaction mixture was then filtered through a celite plug,washed thoroughly with methylene chloride and the filtrate concentratedin vacuo to provide 2-³H-N-desmethoxyapicidin (1.4 mg, 1.8 mgtheoretical, 80%) as a white powder: Analytical RP-HPLC (gradientelution of 40% acetonitrile-water to acetonitrile at 2 mL/min, peakelution at 9.8 min for 2-D-N-desmethoxyapicidin and 10.7 min for2-bromo-N-desmethoxyapicidin Zorbax® Rx-C8, 4.6 mm×25 cm); ¹H NMR(CDCl₃, 500 MHz) satisfactory; ESI-MS (MeCN—H₂O-TFA) m/e 595 (M⁺,C₃₃H₄₆N₅O₅D requires 594.78).

The above step for converting 2-bromo-N-desmethoxyapicidin to2-D-N-desmethoxyapicidin was repeated using ³H₂ to provide the titledtritiated compound.

4. AcGly-Ala-Lys(ε-¹⁴C-Ac)-Arg-His-Arg-Lys(ε-¹⁴C-Ac)-ValNH₂

To a solution of AcGly-Ala-Lys-Arg-His-Arg-Lys-ValNH₂ (0.5 mg, 0.32umol) in N,N-dimethylformamide (DMF, 20 uL) was added anhydrous sodiumcarbonate (5 mg) followed by a solution of [¹⁴C]acetyl chloride indichloromethane (5 uL, 50 uCi, 0.877 umol, specific activity =57mCi/mmol). The mixture was stirred at 25° C. for 0.5 h at which timemethanol (200 uL) was added and the solution filtered through a plug ofglass wool to remove sodium carbonate. The filtrate was concentratedunder nitrogen at 45° C. to near dryness and the resulting residueredissolved in methanol (500 uL). The crude product was purified bypreparative reversed phase HPLC using a VYDAC™ 210HS54 column (9.4×250mm) and a mobile phase consisting of a gradient of 5% to 20%acetonitrile/0.1% aqueous trifluoroacetic acid over 30 min with a flowrate of 3 mL/min. Fractions containing product were combined,concentrated in vacuo to remove acetonitrile, and the pH adjusted to 6.8using 1N sodium bicarbonate solution to provide labeled title compound(17 uCi in 4 mL of aqueous solution, estimated specific activity=114mCi/mmol) which had a radiochemical purity of >99% as determined by HPLCanalysis (VYDAC 210HS54 column, 4.6×250 mm, mobile phase gradient of 5%to 25% acetonitrile/0.1% aqueous trifluoroacetic acid over 20 min, 1mL/min).

5. AcGly-Ala-Lys(ε-³H-Ac)-Arg-His-Arg-Lys(ε-³H-Ac)-ValNH₂

To a solution of AcGly-Ala-Lys-Arg-His-Arg-Lys-ValNH₂ (3.1 mg, 2.0 umol)in 3:2 DMF-water (100 uL) was added sodium [³H]acetate (40 uL of a 0.2 Msolution in 2:1 DMF-water, 8 umol, specific activity=2.9 Ci/mmol)followed by 1-hydroxybenzotriazole hydrate (HOBT, 48 uL of a 0.25 Msolution in DMF, 12 umol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC, 48 uL of a 0.25 M solution in DMF, 12 umol) andN-methylmorpholine (NMM, 48 uL of a 0.5 M solution in DMF, 24 umol). Themixture was let stand at 25° C. overnight and acetic acid (20 uL) andmethanol (180 uL) were added. The product was isolated by HPLC bymultiple injections on a VYDAC 218TP510 column (9.4×250 mm) using amobile phase gradient of 4% to 16% acetonitrile with 0.1% aqueoustrifluoroacetic acid over 16 min with a flow rate or 5 mL/min anddetection at 210 nm. Fractions containing product were combined andconcentrated in vacuo with continuous flushing with ethanol to removeTFA. The labeled title compound (3.7 mCi, estimated specific activity=3Ci/mmol) was submitted as a solution in ethanol (1.5 mL) with aradiochemical purity of 92.5% as determined by HPLC analysis (Zorbax®Rx-C8 column, 4.5×250 mm, 15/85/0.1 methanol: water: perchloroic acid, 1mL/min, 220 nm).

The following non-limiting examples are provided to illustrate theinvention.

EXAMPLE 1 Competitive Binding Assay

(a) Preparation of the Lysates

Chick Liver. Livers from 3-5 three week old chickens are collected,rinsed in ice cold phosphate buffered saline (pH 7.4), diced into smallpieces and rinsed again 3-4 times with ice cold 50 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.4)containing 0.1 mM phenylmethanesulfonyl fluoride (PMSF). The tissue isthen resuspended in HEPES buffer, homogenized in a polytron homogenizerand centrifuged at 3000×g for 10 min. The supernatant is then subjectedto an additional 100,000 g centrifugation for 1 hour. The pellet and thefloating layer of lipid are discarded and the supernatant, referred toas the chick liver S100 fraction, is retained for binding assays. Afterestimating the protein concentration, the supernatant (S100) isaliquoted and frozen at −80 C.

E. tenella oocysts. Approximately 2×10⁹ E. tenella oocysts are suspendedin 5 ml of PBS/0.1 mM PMSF, 4 mls of an equal mixture (vol/vol) of 4.0and 1.0 mm glass beads is added. The glass bead-oocyst mixture is thenshaken for 20 minutes to cause disruption of the oocysts. The efficiencyof breakage is checked microscopically. The resulting homogenate isseparated from the glass beads and centrifuged at 3000×g for 10 min. Thesupernatant is then subjected to an additional centrifugation at 100,000g for 1 hr. The pellet is discarded and the supernatant, referred to asthe E. tenella S100 fraction, is retained for binding assays. Afterestimating the protein concentration, the supernatant (S100) isaliquoted and frozen at −80 C.

P. berghei. 22.5 mls of blood collected from mice infected with P.berghei malaria (with over 50% of the erythrocytes parasitized) iscollected and treated with saponin (1:100 volume of 10% saponin inphosphate buffered saline (PBS)), held on ice for 15 min. to release theparasites and centrifuged at 2000×g for 10 min. The pellet is washedtwice in PBS, and then frozen and thawed 3 times in a total vol of 2 mlPBS containing 0.1 mM PMSF (PBS/PMSF). PBS/PMSF is then added to bringthe volume up to 10 ml and the suspension is centrifuged at 100,000×gfor 1.5 hrs.. The pellet is discarded and the supernatant, refered to asthe P. bergheiS 100 fraction, is retained for binding assays. Afterestimating the protein concentration, the supernatant (S100) isaliquoted and frozen at −80 C.

(b) Competitive Binding Assay

Each assay tube contains a final volume of 1 ml with 1.3 ng/ml of³H-N-desmethoxyapicidin, 150-200 μg of the appropriate S100 supernatantand concentrations of potential HDA inhibitors from 0.001-10 μg/ml.

(i) labeled substrate. 2-³H-N-desmethoxyapicidin (18.69 mCi/mg) isdiluted 1:100 in ethanol, and 2 μl added per 1 ml of 50 mM HEPES pH 7.4with 0.05% Triton-X100 (HEPES/Triton®) is added. This provides 1.3 ng2-³H-N-desmethoxyapicidin /ml of assay (54,000 DPM/ml.)

(ii) potential HDA inhibitors. The stocks of the test compounds are madeup to 2 mg/ml dimethylsulfoxide (DMSO). The stock solution is diluted to1000, 100, 10, 1 and 0.1 μg/ml in ethanol where a full titration isrequired. Ethanol is used as a negative control, a 100 μg/ml solution ofunlabeled N-desmethoxyapicidin is used as a positive control. Six μl ofthe test compound solution, the positive and negative controls are usedfor the assay.

(iii) supernatant from (a). The S100 sample is diluted to aconcentration of 3.0 mg protein/ml (chick liver) or 4 mg protein/ml(parasite) in the HEPES/Triton® buffer. Fifty μl is used for the assay.

Stock solutions of potential HDA inhibitors (ii) are diluted into 50 mMHEPES/Triton® buffer to achieve a final concentration of test compoundin the range of 0.001-10 μM. ³H-N-desmethoxyapicidin is added to a finalconcentration of 1.3 ng/ml. 50 μl of parasite or chick liver S100solution (150-200 μg) is added to each tube. The assay mixture isincubated for 1 hr at 25° C. and then aspirated through Whatman GF/Bfilters impregnated with polyethylenimine (filters prepared in advanceby soaking in 2 ml of polyethylenimine per 330 mls water at room tempfor 1 hour). Assay tubes were rinsed 3 times with 1-2 mls ofHEPES/Triton and aspirated through the same filter. The filter is thendried and counted in scintillant.

Compounds Ia, Ib, Ic, IIA, IIB, trichostatin, trichostatic acid, andHC-toxin were evaluated in the binding assay using E. tenella andchicken liver lysates. Trichostatic acid showed IC₅₀ values of >1000 nMin both E. tenella and chicken liver lysates. All the other compoundsshowed IC₅₀ values in E. tenella lysate ranging from 4-60 nM, and exceptfor compounds IIA and IIB, they showed IC₅₀ values in E. tenella lysateranging from 2-35 nM. Compounds IIA and IIB are selective for E. tenellaover chicken liver lysate (IC₅₀ in chicken liver >1000 nM). Theantiparasitic activity of compounds Ia, Ib, Ic, trichostatin,trichostatic acid, HC-toxin were evaluated in vitro against E. tenella(1) and P. falciparum. (2) Trichostatic acid, which showed IC₅₀ valuesof >1000 nM in both E. tenella and chicken liver lysates, was not activeagainst either parasite; the other compounds have minimum inhibitoryconcentrations ranging from 8 to 250 ng/ml against E. tenella, and IC₅₀ranging from 7 to 115 ng/ml against P. falciparum.

(1) Generally following the procedure described in Crane, Schmatz,Stevens, Habbersett and Murray. Eimeria tenella: in vitro development inirradiated bovine kidney cells.(1984) Parasitology 88: 521-530.

(2) Generally following the procedure described in Desjardins et al,“Quantitative Assessment of Antimalarial Activity In Vitro by aSemiautomated Microdilution Technique” Antimicrobial Agents andChemotherapy 1979, 16:710-718.

EXAMPLE 2 Determination of Histone Acetylation States

Preparation of HeLa Cell Histones

About 2×10⁷ HeLa cells growing at 37 C were treated with variouscompounds in the standard tissue culture medium (MEM containing 10%fetal calf serum) for 4 hrs. Cells were then removed from the flask byscraping in PBS in a total volume of about 10 mls. The cells were washedtwice in PBS and resuspended in about 3 mls lysis buffer (Lysis buffer;0.45% Nonidet® P-40, 10 mM tris, 10 mM NaCl, 5 mM MgCl, 0.1 mM EGTA, 0.1mM PMSF). The suspension was mixed by vortexing for 10 seconds,incubated on ice for 15 minutes and nuclei were pelleted bycentrifugation through a 1 ml cushion of 30% sucrose in lysis buffer at1300×g for 10 minutes. The supernatant was discarded and the nuclei werethen resuspended in 1.0 ml water. H₂SO₄ was added to a finalconcentration of 0.4M and the nuclear suspension was incubated on icefor 30 min. and then pelleted by centrifugation at 13,000×g for 10minutes. The pellets were discarded, and ten volumes of ice cold acetonewas added to the supernatant proteins and left overnight at −20° C. toprecipitate soluble proteins. These proteins (predominantly histones)were pelleted by centrifugation at 10,000×g for 15 minutes at 4° C. andresuspended in AUT sample buffer (Sample buffer: 0.9M acetic acid, 0.02%w/v methyl green) in preparation for gel electrophoresis.

Gel Electrophoresis

Acid urea triton (AUT) polyacrylamide gels were performed according tothe methods of Alfageme et al (1974) J. Biol. Chem. 249, 3729, withmodifications as described in Lennox and Cohen (Methods in Enzymology,170, 532-549). For optimal resolution of HeLa cell and Plasmodiumfalciparum histones the separation gel contained 7.5M urea, 12%acrylamide, 0.38% Triton® X 100, 0.08% bis acrylamide, and 0.87M aceticacid. The loading gels contained 7.5M urea, 0.37% Triton X 100, 6%acrylamide, 0.04% bis acrylamide and 0.87M acetic acid. Gels were run in1M acetic acid. Gels were pre-electrophoresed at 350V for 1 hour and runat 450V for 3.5 hours. Gels were either stained with Coomassie BrilliantBlue R in 7% acetic acid, 20% methanol and then destained in 7% aceticacid and 20% methanol, or treated with Enlightening (from New EnglandNuclear), dried and radiolabel detected by fluorography.

As shown in FIG. 1. apicidin, trichostatin and HC-toxin cause a distinctshift in the histone gel profiles relative to untreated cells. Thechanges were most distinct for H4 histones. Due to the hyperacetylationof the histones in the treated cells there is a distinct increase in thehigher bands of H4 and a decrease in the lower bands for H4. This occursbecause the higher the acetylation state of the histones the slower theymigrate in the gel.

Labeling P. falciparum with ¹⁴C acetate

Approximately 1×10⁹ P. falciparum infected red blood cells (about 60%parasitized) were resuspended in 10 ml of RPMI 1640 containing variousconcentrations of test compounds and 250 μCi ¹⁴C sodium acetate andincubated at 37° C. for 2 hours. The cells were then washed 3 time withPBS and histones were prepared as described below.

Preparation of P. falciparumHistones

Histones were prepared according to the method of Cary et al (1994)Parasitology Research, 80, 255-258. P. falciparum infected red bloodcells were lysed by the addition of 1/100th volume of 10% saponin inPBS, and the released parasites were washed with PBS and pelleted.Parasites were resuspended in 1 ml hypotonic lysis buffer (10 mM tris pH8.0, 1.5mM MgCl, 2mM PMSF, 0.2mM N-α-p-tosyl-L-lysine chloromethylketone (TLCK), 0.25 μg/ml leupeptin and 10 μg/ml pepstatin), incubatedon ice for 15 minutes and lysed with 100 strokes in a douncehomogenizer. A nuclei enriched pellet was obtained by centrifugation at13,000×g for 10 minutes, the pellet was resuspended in water andhistones were prepared and run on AUT gels as described for HeLa cells.

As shown in FIG. 2 both apicidin and trichostatin cause hyperacetylationof P. falciparum histones. This is indicated by the overall increase inthe amount of 14C-acetylation and a shift in the pattern of the labeledhistone bands. The upward shift in treated parasites occurs due to anincrease in the population of histones with many acetylated lysines(which migrate slower in the gel) and a decrease in the number ofnon-acetylated or minimally acetylated (which migrate faster in thegel).

EXAMPLE 3 Histone Deacetylase Inhibition Assay (all temperatures in °C.)

Assay 1 for Histone Deacetylase Activity and Inhibition

The standard assay contained in a total volume of 40 μL: 400 nmolHEPES-sodium, pH 7.4, 100 pmol of the substrate[AcGly-Ala-Lys(ε-¹⁴C-Ac)-Arg-His-Arg-Lys(ε-¹⁴C-Ac)-ValNH₂] (see Kervabonet al (1979) FEBS Letters 106: 93-96) having a specific activity ofapprox. 114 mCi/mmol, and a source of histone deacetylase (HDAase)activity. The amount of HDAase added was chosen such that ≦20% of thesubstrate was consumed during the assay. The reaction was initiated byenzyme addition and allowed to proceed for 60 min at 41°. At 60 min, thereaction was terminated by the addition of a 50% slurry of Amberlite® AG50W×4 cation exchange resin, sodium form (200-400 mesh) in 25 mM sodiumacetate buffer, pH 4.2 (200 μL). The resin binds both remainingsubstrate and the (partially) deacetylated peptidyl products. Thequenched reaction was then incubated for at least 30 min at 25° withoccasional mixing, diluted with additional 25 mM sodium acetate buffer,pH 4.2 (760 μL; final volume 1000 μL), incubated for a minimum of anadditional 30 min at 25° with occasional mixing, and then centrifuged at10,000×g for 1 min. An aliquot of the supernatant (800 μL) containingthe enzymatically released ¹⁴C-acetate was removed, mixed with Aquasol 2liquid scintillation counter (LSC) cocktail (10 mL), and counted in aBeckman model LS-5801 LSC. To assure that the acetate released was duespecifically to the action of HDAase, a parallel control incubation wasperformed which contained a known HDAase inhibitor [originally, 1-5 mMbutyrate (see Cousens et al (1979) J. Biol. Chem. 254: 1716-1723);later, 40-1000 nM apicidin in DMSO once it had been demonstrated to bean HDAase inhibitor]; the amount of radioactivity generated in thepresence of inhibitor was subtracted from the value obtained in theabsence of inhibitor in order to calculate HDAase dependent acetateproduction.

For inhibition studies, the inhibitor under examination was added to thestandard assay cocktail at the desired concentration in dimethylsulfoxide (final concentration of DMSO in the reaction was kept constantat 2.5% v/v) and the HDAase activity compared to that found in control(minus inhibitor) incubations which lacked inhibitor but contained 2.5%v/v final DMSO.

Assay 2 for Histone Deacetylase Activity and Inhibition

The standard assay contained in a total volume of 200 μL: 2000 nmolHEPES-sodium, pH 7.4, 11 pmolAcGly-Ala-Lys(ε-³H-Ac)-Arg-His-Arg-Lys(ε-³H-Ac)-ValNH₂ having a specificactivity of approximately 3 Ci/mmol, and a source of histone deacetylase(HDAase) activity. The amount of HDAase added was chosen such that ≦20%of the substrate was consumed during the assay. The reaction wasinitiated by enzyme addition and allowed to proceed for 60 min at 410.At 60 min, the reaction was terminated by the addition of a aqueoussolution containing 0.1 M acetic acid and 0.5 M hydrochloric acid (20μL), followed by the addition of ethyl acetate (1000 μL). The quenchedreaction was then vortexed for at least 15 sec at 25° and thencentrifuged at 10,000×g for 1 min. An aliquot of the ethyl acetate phase(900 μL) containing the enzymatically released ³H-acetate was removed,mixed with Aquasol 2 liquid scintillation counter (LSC) cocktail (6 mL),and counted in a Beckman model LS-5801 LSC. To assure that the acetatereleased was due specifically to the action of HDAase, a parallelcontrol incubation was performed which contained a known HDAaseinhibitor [originally, 1-5 mM butyrate; later, 40-1000 nM apicidin inDMSO once it had been demonstrated to be an HDAase inhibitor]; theamount of radioactivity generated in the presence of inhibitor wassubtracted from the value obtained in the absence of inhibitor in orderto calculate HDAase dependent acetate production.

For inhibition studies, the inhibitor under examination was added to thestandard assay cocktail at the desired concentration in dimethylsulfoxide (final concentration of DMSO in the reaction was kept constantat 0.5% v/v) and the HDAase activity compared to that found in control(minus inhibitor) incubations which lacked inhibitor but contained 0.5%v/v final DMSO.

Preparation of Hela Nuclear Extract as a Source of HDAase

Hela nuclei were prepared by the method of Cousens et al, supra. Theresulting nuclear pellet was hypotonically lyzed by the addition of ˜4volumes of cold 25 mM HEPES-sodium, pH 7.4 and incubation at 4° C. for10 min with intermittant vortexing. The suspension was then centrifugedat 10,000×g for 10 min at 4° and the resulting supernatant used as asource of HDAase activity.

Preparation of Chicken Red Blood Cell Nuclear Extract as a Source ofHDAase

Chickens were sacrificed by asphyxiation with carbon dioxide,decapitated, and bled into a beaker containing one tenth volume ofanticoagulant solution (16 mM citric acid, 89 mM trisodium citrate, 16mM sodium dihydrogen phosphate, 130 mM glucose) on ice. The blood wasfiltered through four layers of cheesecloth and the erythrocytessedimented (1500 g, 5 min, 4°) and the supernatant discarded. Theerythrocytes were washed twice by suspension in three volumes of bufferA [10 mM Tris chloride, pH 7.2, 50 mM sodium bisulfite, 10 mM magnesiumchloride, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 8.6% w/vsucrose] and subsequent centrifugation (1500 g, 5 min, 4°). The washederythrocytes were suspended in an equal volume of buffer A, onehundredth volume of 10% v/v saponin added (final concentration, 0.1%v/v), and the suspension incubated for ˜20 min with occasion gentleswirling until microscopic examination revealed that >95% of the cellshad been lyzed. The suspension was centrifuged (1500 g, 5 min, 4°) topellet unlyzed cells. The resulting supernatants were carefully pouredoff, diluted with additional buffer A (two volumes, final of buffer A tostarting erythrocytes, 3:1), centrifuged (3000 g, 45 min, 4°), and thesupernatants discarded. The resulting nuclear pellets were washed twiceby suspension in three volumes of buffer A and subsequent centrifugation(3000 g, 45 min, 4°) followed by a third wash with three volumes ofbuffer A lacking sodium bisulfite and PMSF. The resulting nuclear pelletwas hypotonically lyzed by the addition of ˜4 volumes of cold 25 mMHEPES-sodium, pH 7.4 and incubation at 4° for 10 min with intermittantvortexing. The suspension was then centrifuged at 10,000×g for 10 min at4° and the resulting supernatant used as a source of HDAase activity.

Preparation of Eimeria tenella Extract as a Source of HDAase

Preparation of the S100 extract from Eimeria tenella sporulated oocystswas as described above. A portion (2.5 mL) of the extract wasconcentrated five fold using a centricon ultrafiltration device and theresulting concentrate (500 μL) chromatographed on a Superose 12 HR 10/30gel filtration column equilibrated with 25 mM HEPES-sodium, pH 7.4.Fractions (500 μL) containing apicidin inhibitable HDAase activity(18-23) were pooled and utilized as the source of Eimeria HDAaseactivity.

Correlation between HDA Catalytic Activity and Apicidin Binding

When total chick liver homogenate was subjected to ion exchangechromatography an apicidin and butyrate inhibitable HDAse activitycoeluted with the 2-³H-N-desmethoxyapicidin binding activity. Partialpurification of chicken red blood cell nuclear HDAase by anion exchangechromatography and gel filtration also resulted in coelution of theenzyme activity and the binding activity. Moreover, homogenates ofEimeria tenella sporulated oocysts subjected to ammonium sulfateprecipitation and gel filtration resulted in the partial purification ofa butyrate and apicidin inhibitable Eimeria HDAase which coeluted with2-³H-N-desmethoxyapicidin binding activity on the column. These datastrongly suggested that the binding and enzyme activity were directlyrelated in both host and parasite, and that in fact the target of³H-N-desmethoxyapicidin binding was HDAase. Apicidin, trichostatin,trichostatic acid, HC-toxin and n-butyrate were evaluated in the histonedeacetylase inhibition assay (Assay 1), and the results correlate withthat obtained in the enzyme and ³H-N-desmethoxyapicidin competitivebinding assays. Thus, trichostatic acid, which showed no activity in thebinding assay, is also inactive in the enzyme inhibition assay. Theother compounds showed IC₅₀ values in the range of 2-20 nM in the enzymeinhibition assay with E. tenella and chicken liver extracts (with theexception of n-butyrate which had IC₅₀ values consistent with thosereported in the literature). Consequently, a binding assay using aninhibitor or substrate of histone deacetylase as the ligand could beused to identify and design antiprotozoal agents.

What is claimed is:
 1. A method for identifying compounds that areantiprotozoal agents by determining whether a test compound or naturalproduct extract inhibits the action of protozoal histone deacetylase,said method comprising: (a) contacting a protozoal histone deacetylase,or an extract containing protozoal histone deacetylase with a knownamount of a labeled compound that interacts with a protozoal histonedeacetylase; in the presence or absence of a known dilution of a testcompound or a natural product extract; and (b) quantitating the percentinhibition of interaction of said labeled compound by the difference inthe enzyme activities in the presence and absence of the inhibitor testcompound or natural product extract.
 2. The method of claim 1 whereinsaid labeled compound binds to histone deacetylase.
 3. The method ofclaim 1 wherein said labeled compound is a substrate of histonedeacetylase.